Compositions and methods for treatment of mitochondrial diseases

ABSTRACT

Compounds, compositions, and methods are provided for treatment of disorders related to mitochondrial dysfunction. The methods comprise administering to a mammal a composition containing pyrimidine nucleotide precursors in amounts sufficient to treat symptoms resulting from mitochondrial respiratory chain deficiencies.

FIELD OF THE INVENTION

[0001] This invention relates generally to compounds and methods fortreatment and prevention of diseases, developmental delays, and symptomsrelated to mitochondrial dysfunction. Pyrimidine nucleotide precursorsare administered to a mammal, including a human, for the purpose ofcompensating for mitochondrial dysfunction and for improvingmitochondrial functions.

BACKGROUND OF THE INVENTION

[0002] Mitochondria are cellular organelles present in most eukaryoticcells. One of their primary functions is oxidative phosphorylation, aprocess through which energy derived from metabolism of fuels likeglucose or fatty acids is converted to ATP, which is then used to drivevarious energy-requiring biosynthetic reactions and other metabolicactivities. Mitochondria have their own genomes, separate from nuclearDNA, comprising rings of DNA with about 16,000 base pairs in humancells. Each mitochondrion may have multiple copies of its genome, andindividual cells may have hundreds of mitochondria.

[0003] Mitochondrial dysfunction contributes to various disease states.Some mitochondrial diseases are due to mutations or deletions in themitochondrial genome. Mitochondria divide and proliferate with a fasterturnover rate than their host cells, and their replication is undercontrol of the nuclear genome. If a threshold proportion of mitochondriain a cell is defective, and if a threshold proportion of such cellswithin a tissue have defective mitochondria, symptoms of tissue or organdysfunction can result. Practically any tissue can be affected, and alarge variety of symptoms may be present, depending on the extent towhich different tissues are involved.

[0004] A fertilized ovum might contain both normal and geneticallydefective mitochondria. The segregation of defective mitochondria intodifferent tissues during division of this ovum is a stochastic process,as will be the ratio of defective to normal mitochondria within a giventissue or cell (although there can be positive or negative selection fordefective mitochondrial genomes during mitochondrial turnover withincells). Thus, a variety of different pathologic phenotypes can emergeout of a particular point mutation in mitochondrial DNA. Conversely,similar phenotypes can emerge from mutations or deletions affectingdifferent genes within mitochondrial DNA. Clinical symptoms incongenital mitochondrial diseases often manifest in postmitotic tissueswith high energy demands like brain, muscle, optic nerve, andmyocardium, but other tissues including endocrine glands, liver,gastrointestinal tract, kidney, and hematopoietic tissue are alsoinvolved, again depending in part on the segregation of mitochondriaduring development, and on the dynamics of mitochondrial turnover overtime.

[0005] In addition to congenital disorders involving inherited defectivemitochondria, acquired mitochondrial dysfunction contributes todiseases, particularly neurodegenerative disorders associated with aginglike Parkinson's, Alzheimer's, Huntington's Diseases. The incidence ofsomatic mutations in mitochondrial DNA rises exponentially with age;diminished respiratory chain activity is found universally in agingpeople. Mitochondrial dysfunction is also implicated in excitotoxicneuronal injury, such as that associated with seizures or ischemia.

[0006] Treatment of diseases involving mitochondrial dysfunction hasheretofore involved administration of vitamins and cofactors used byparticular elements of the mitochondrial respiratory chain. Coenzyme Q(ubiquinone), nicotinamide, riboflavin, carnitine, biotin, and lipoicacid are used in patients with mitochondrial disease, with occasionalbenefit, especially in disorders directly stemming from primarydeficiencies of one of these cofactors. However, while useful inisolated cases, no such metabolic cofactors or vitamins have been shownto have general utility in clinical practice in treating mitochondrialdiseases. Similarly, dichloracetic acid (DCA) has been used to treatmitochondrial cytopathies such as MELAS; DCA inhibits lactate formationand is primarily useful in cases of mitochondrial diseases whereexcessive lactate accumulation itself is contributing to symptoms.However, DCA does not address symptoms related to mitochondrialinsufficiency per se and can be toxic to some patients, depending on theunderlying molecular defects.

[0007] Mitochondrial diseases comprise disorders caused by a hugevariety of molecular lesions or defects, with the phenotypic expressionof disease further complicated by stochastic distributions of defectivemitochondria in different tissues.

[0008] Commonly owned U.S. Pat. No. 5,583,117 discloses acylatedderivatives of cytidine and uridine. Commonly owned application PCT/US96/10067 discloses the use of acylated pyrimidine nucleosides to reducethe toxicity of chemotherapeutic and antiviral pyrimidine nucleosideanalogs.

OBJECTS OF THE INVENTION

[0009] It is an object of the invention to provide compositions andmethods for treating disorders or pathophysiology associated withmitochondrial dysfunction or mitochondrial respiratory chain dysfunctionin a mammal, including a human.

[0010] It is an object of the invention to provide compounds andcompositions that improve tissue resistance to mitochondrial dysfunctionin vivo.

[0011] It is an object of the invention to provide compositions andmethods for treatment of mitochondrial diseases.

[0012] It is an object of the invention to provide agents whichcompensate broadly for mitochondrial deficits involving a wide varietyof molecular pathologies, since, in many cases, precise diagnosis ofmolecular lesions in mitochondrial disorders is difficult.

[0013] It is an object of the invention to provide a practical treatmentfor mitochondrial diseases that is beneficial in the case ofmitochondrial electron transport chain deficits regardless of thespecific molecular defects.

[0014] It is an object of the invention to provide not only for therelatively rare congenital diseases related to mitochondrial DNAdefects, but also for significant neuromuscular and neurodevelopmentaldisorders that appear in childhood and for common age-relateddegenerative diseases like Alzheimer's or Parkinson's Diseases.

[0015] It is an object of the invention to provide compositions andmethods for treatment and prevention of neurodegenerative andneuromuscular disorders.

[0016] It is an object of the invention to provide compositions andmethods for treatment and prevention of epilepsy.

[0017] It is an object of the invention to provide compositions andmethods for treatment and prevention of migraine.

[0018] It is an object of the invention to provide compositions andmethods for preventing death or dysfunction of postmitotic cells in amammal, including a human.

[0019] It is an object of the invention to provide compositions andmethods for treatment of neurodevelopmental delay disorders

[0020] It is a further object of the invention to provide a compositionfor treatment or prevention of tissue damage due to hypoxia or ischemia.

[0021] It is a further object of this invention to provide compositionsand methods for treating or preventing ovarian dysfunction, menopause,or secondary consequences of menopause.

[0022] It is a further object of the invention to provide compositionsand methods for reducing side effects of cancer chemotherapies due tochemotherapy-induced mitochondrial injury.

[0023] It is a further object of the invention to provide a method fordiagnosing mitochondrial disease and dysfunction.

SUMMARY OF THE INVENTION

[0024] The subject invention provides a method for treatingpathophysiological consequences of mitochondrial respiratory chaindeficiency in a mammal comprising administering to such a mammal in needof such treatment an amount of a pyrimidine nucleotide precursoreffective in reducing the pathophysiological consequences. Additionally,the invention provides a method of preventing pathophysiologicalconsequences of mitochondrial respiratory chain deficiency comprisingadministering to a mammal an amount of a pyrimidine nucleotide precursoreffective in preventing the pathophysiological consequences.

[0025] In mitochondrial disease the compounds and compositions of theinvention are useful for attenuating clinical sequelae stemming fromrespiratory chain deficiencies. Respiratory chain deficienciesunderlying mitochondrial disease are caused by various factors includingcongenital or inherited mutations and deletions in mitochondrial DNA,deficits in nuclear-encoded proteins affecting respiratory chainactivity, as well as somatic mutations, elevated intracellular calcium,excitotoxicity, nitric oxide, hypoxia and axonal transport defects.

[0026] The subject invention provides compounds, compositions, andmethods for preventing or reducing death and dysfunction of postmitoticcells bearing mitochondrial respiratory chain deficits.

[0027] The subject invention furthermore provides compounds,compositions, and methods for treating neurodevelopmental delays inlanguage, motor, executive function, cognitive, and neuropsychologicalsocial skills.

[0028] The subject invention also relates to treatment of disorders andconditions that are herein disclosed as conditions to whichmitochondrial defects contribute and which therefore are subject totreatment with compounds, and compositions of the invention. Theseinclude side effects of cancer chemotherapy like peripheralneuropathies, nephropathies, fatigue, and early menopause, as well asovulatory abnormalities and normal menopause itself.

[0029] The subject invention also relates to a method for diagnosingmitochondrial diseases by treating patients with a pyrimidine nucleotideprecursor and assessing clinical benefit in selected signs and symptoms.

[0030] The invention, as well as other objects, features and advantagesthereof will be understood more clearly and fully from the followingdetailed description, when read with reference to the accompanyingresults of the experiments discussed in the examples below.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The subject invention is related to compounds, compositions, andmethods for treating or preventing a variety of clinical disorderssecondary to mitochondrial dysfunction, especially deficits in theactivity of components of the mitochondrial respiratory chain. Suchdisorders include congenital mitochondrial cytopathies,neurodevelopmental delays, age-related neurodegenerative diseases, aswell as particular diseases affecting the heart, peripheral andautonomic nerves, skeletal muscle, pancreas and other tissues andorgans.

A. Definitions

[0032] “Mitochondrial disease” refers to disorders to which deficits inmitochondrial respiratory chain activity contribute in the developmentof pathophysiology of such disorders in a mammal. This categoryincludes 1) congenital genetic deficiencies in activity of one or morecomponents of the mitochondrial respiratory chain; 2) acquireddeficiencies in the activity of one or more components of themitochondrial respiratory chain, wherein such deficiencies are causedby, inter alia, a) oxidative damage during aging; b) elevatedintracellular calcium; c) exposure of affected cells to nitric oxide; d)hypoxia or ischemia; or e) microtubule-associated deficits in axonaltransport of mitochondria.

[0033] The mitochondrial respiratory chain (also known as the electrontransport chain) comprises 5 major complexes:

[0034] Complex I NADH:ubiquinone reductase

[0035] Complex II Succinate:ubiquinone reductase

[0036] Complex III ubiquinol:cytochrome-c reductase

[0037] Complex IV cytochrome-c oxidase

[0038] Complex V ATP synthase

[0039] Complexes I and II accomplish the transfer of electrons frommetabolic fuels like glycolysis products and fatty acids to ubiquinone(Coenzyme Q), converting it to ubiquinol. Ubiquinol is converted back toubiquinone by transfer of electrons to cytochrome c in Complex III.Cytochrome c is reoxidized at Complex IV by transfer of electrons tomolecular oxygen, producing water. Complex V utilizes potential energyfrom the proton gradient produced across the mitochondrial membrane bythese electron transfers into ATP.

[0040] Dihydro-orotate dehydrogenase (DHODH), is an enzyme involved inde novo synthesis of uridine nucleotides. DHODH activity is coupled tothe respiratory chain via transfer of electrons from dihydro-orotate toubiquinone; these electrons are then passed on to cytochrome c andoxygen via Complexes III and IV respectively. Only Complexes III and IVare directly involved in pyrimidine biosynthesis. Orotate produced bythe action of DHODH is converted to uridine monophosphate byphosphoribosylation and decarboxylation.

[0041] “Pyrimidine nucleotide precursors” in the context of theinvention are intermediates in either the de novo or salvage pathways ofpyrimidine nucleotide synthesis that enter into pyrimidine synthesiseither distal to DHODH (e.g. orotate) or which do not require DHODHactivity for conversion to pyrimidine nucleotides (e.g. cytidine,uridine, or acyl derivatives of cytidine or uridine). Also includedwithin the scope of the invention are pyrimidine nucleoside phosphates(e.g. nucleotides, cytidine diphosphocholine, uridine diphosphoglucose);these compounds are degraded to the level of uridine or cytidine priorto entry into cells and anabolism. Acyl derivatives of cytidine anduridine have better oral bioavailability than the parent nucleosides ornucleotides. Orotic acid and esters thereof are converted to uridinenucleotides and are also useful for accomplishing the goals of theinvention.

B. Compounds of the Invention

[0042] A primary feature of the present invention is the unexpecteddiscovery that administration of pyrimidine nucleotide precursors iseffective in treatment of a large variety of symptoms and disease statesrelated to mitochondrial dysfunction.

[0043] Tissue pyrimidine nucleotide levels are increased byadministration of any of several precursors. Uridine and cytidine areincorporated into cellular nucleotide pools by phosphorylation at the 5′position; cytidine and uridine nucleotides are interconvertible throughenzymatic amination and de-amination reactions. Orotic acid is a keyintermediate in de novo biosynthesis of pyrimidine nucleotides.Incorporation of orotic acid into nucleotide pools requires cellularphosphoribosyl pyrophosphate (PRPP). Alternatively (or in addition toprovision of exogenous nucleotide precursors), availability of uridineto tissues is increased by administration of compounds which inhibituridine phosphorylase, the first enzyme in the pathway for degradationof uridine. The compounds of the invention useful in treatingmitochondrial diseases and related disorders include uridine, cytidine,orotate, orally bioavailable acyl derivatives or esters of thesepyrimidine nucleotide precursors, and inhibitors of the enzyme uridinephosphorylase.

[0044] In reference to acyl derivatives of cytidine and uridine, thefollowing definitions pertain:

[0045] The term “acyl derivative” as used herein means a derivative of apyrimidine nucleoside in which a substantially nontoxic organic acylsubstituent derived from a carboxylic acid is attached to one or more ofthe free hydroxyl groups of the ribose moiety of the oxypurinenucleoside with an ester linkage and/or where such a substituent isattached to the amine substituent on the purine ring of cytidine, withan amide linkage. Such acyl substituents are derived from carboxylicacids which include, but are not limited to, compounds selected from thegroup consisting of a fatty acid, an amino acid, nicotinic acid,dicarboxylic acids, lactic acid, p-aminobenzoic acid and orotic acid.Advantageous acyl substituents are compounds which are normally presentin the body, either as dietary constituents or as intermediarymetabolites.

[0046] The term “pharmaceutically acceptable salts” as used herein meanssalts with pharmaceutically acceptable acid or base addition salts ofthe derivatives, which include, but are not limited to, sulfuric,hydrochloric, or phosphoric acids, or, in the case of orotate, sodium orcalcium hydroxides, and cationic amino acids, especially lysine.

[0047] The term “amino acids” as used herein includes, but is notlimited to, glycine, the L forms of alanine, valine, leucine,isoleucine, phenylalanine, tyrosine, proline, hydroxyproline, serine,threonine, cysteine, cystine, methionine, tryptophan, aspartic acid,glutamic acid, arginine, lysine, histidine, ornithine, hydroxylysine,carnitine, and other naturally occurring amino acids.

[0048] The term “fatty acids” as used herein means aliphatic carboxylicacids having 2-22 carbon atoms. Such fatty acids may be saturated,partially saturated or polyunsaturated.

[0049] The term “dicarboxylic acids” as used herein means fatty acidswith a second carboxylic acid substituent.

[0050] Compounds of the invention have the following structures:

[0051] In all cases except where indicated, letters and letters withsubscripts symbolizing variable substituents in the chemical structuresof the compounds of the invention are applicable only to the structureimmediately preceding the description of the symbol.

[0052] (1) An acyl derivative of uridine having the formula:

[0053] wherein R₁, R₂, R₃ and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite, provided that at least oneof said R substituents is not hydrogen, or a pharmaceutically acceptablesalt thereof.

[0054] (2) An acyl derivative of cytidine having the formula:

[0055] wherein R₁, R₂, R₃ and R₄ are the same or different and each ishydrogen or an acyl radical of a metabolite, provided that at least oneof said R substituents is not hydrogen, or a pharmaceutically acceptablesalt thereof.

[0056] The compounds of the invention useful in treating mitochondrialdiseases include:

[0057] (3) An acyl derivative of uridine having the formula:

[0058] wherein R₁, R₂, and R₃ are the same, or different, and each ishydrogen or an acyl radical of

[0059] a. an unbranched fatty acid with 2 to 22 carbon atoms,

[0060] b. an amino acid selected from the group consisting of glycine,the L forms of alanine, valine, leucine, isoleucine, tyrosine, proline,hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid,glutamic acid, arginine, lysine, histidine, carnitine and ornithine,

[0061] c. a dicarboxylic acid having 3-22 carbon atoms,

[0062] d. a carboxylic acid selected from one or more of the groupconsisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvicacid, lipoic acid, pantothenic acid, acetoacetic acid, p-aminobenzoicacid, betahydroxybutyric acid, orotic acid, and creatine.

[0063] (4) An acyl derivatives of cytidine having the formula:

[0064] wherein R₁, R₂, R₃, and R₄ are the same, or different, and eachis hydrogen or an acyl radical of

[0065] a. an unbranched fatty acid with 2 to 22 carbon atoms,

[0066] b. an amino acid selected from the group consisting of glycine,the L forms of phenylalanine, alanine, valine, leucine, isoleucine,tyrosine, proline, hydroxyproline, serine, threonine, cystine, cysteine,aspartic acid, glutamic acid, arginine, lysine, histidine carnitine andornithine,

[0067] c. a dicarboxylic acid having 3-22 carbon atoms,

[0068] d. a carboxylic acid selected from one or more of the groupconsisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvicacid, lipoic acid, pantothenic acid, acetoacetic acid, p-aminobenzoicacid, betahydroxybutyric acid, orotic acid, and creatine.

[0069] (5) An acyl derivative of uridine having the formula:

[0070] wherein at least one of R₁, R₂, or R₃ is a hydrocarbyloxycarbonylmoiety containing 2-26 carbon atoms and the remaining R substituents areindependently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety orH or phosphate.

[0071] (6) An acyl derivative of cytidine having the formula:

[0072] wherein at least one of R₁, R₂, R₃ or R₄ is ahydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and theremaining R substituents are independently a hydrocarbyloxycarbonyl orhydrocarbylcarbonyl moiety or H or phosphate.

[0073] (7) Orotic acid or salts thereof:

[0074] Pharmaceutically-acceptable salts of orotic acid include those inwhich the cationic component of the salt is sodium, potassium, a basicamino acid such as arginine or lysine, methylglucamine, choline, or anyother substantially nontoxic water soluble cation with a molecularweight less than about 1000 daltons.

[0075] 8) Alcohol-substituted orotate derivatives:

[0076] wherein R₁ is a radical of an alcohol containing 1 to 20 carbonatoms joined to orotate via an ester linkage.

[0077] Also encompassed by the invention are the pharmaceuticallyacceptable salts of the above-noted compounds.

[0078] Advantageous compounds of the invention are short-chain (2 to 6carbon atoms) fatty acid esters of uridine or cytidine. Particularlyadvantageous compounds are triacetyluridine or triacetylcytidine. Suchcompounds have better oral bioavailabilty than the parent nucleosides,and are rapidly deacetylated following absorption after oraladministration.

[0079] Pyruvic acid is useful for treatment of cells with defectivemitochondrial function. Cells with reduced capability for mitochondrialoxidative phosphorylation must rely on glycolysis for generation of ATP.Glycolysis is regulated by the redox state of cells. Specifically, NAD+is required for optimal glucose flux, producing NADH in the process. Inorder to maximize energy production from glycolysis, NADH must bereoxidized to NAD+. Exogenous pyruvate can reoxidize NADH, in part via aplasma membrane enzyme, NADH Oxidase.

[0080] Uridine tripyruvate (2′,3′,5′-tri-O-pyruvyluridine) provides thebenefits of both pyrimidines and pyruvate, delivering both with a singlechemical entity, and avoiding the load of sodium, calcium, or othercations in the corresponding salts of pyruvic acid.

Inhibitors of Uridine Phosphorylase

[0081] An alternative or complementary strategy for treatingmitochondrial diseases involves inhibition of uridine catabolism with aninhibitor of the enzyme uridine phosphorylase.

[0082] Examples of inhibitors of uridine phosphorylase that are usefulfor treatment of mitochondrial disease include but are not limited to5-benzyl barbiturate or 5-benzylidene barbiturate derivatives including5-benzyl barbiturate, 5-benzyloxybenzyl barbiturate,5-benzyloxybenzyl-1-[(1 -hydroxy-2-ethoxy)methyl] barbiturate,5-benzyloxybenzylacetyl- 1-[(1-hydroxy-2-ethoxy)methyl] barbiturate, and5-methoxybenzylacetylacyclobarbiturate, 2,2′-anhydro-5-ethyluridine,5-ethyl-2-deoxyuridine and acyclouridine compounds, particularly5-benzyl substituted acyclouridine congeners including but not limitedto benzylacyclouridine, benzyloxybenzylacyclouridine,aminomethyl-benzylacyclouridine,aminomethylbenzyloxy-benzylacyclouridine,hydroxymethyl-benzylacyclouridine, andhydroxymethyl-benzyloxy-benzylacyclouridine. See also WO 89/09603 and WO91/16315, hereby incorporated by reference.

C. Compositions of the Invention

[0083] In one embodiment of the invention, novel pharmaceuticalcompositions comprise as an active agent one or more pyrimidinenucleotide precursors selected from the group consisting of uridine,cytidine, orotic acid or its salts or esters, and acyl derivatives ofthese pyrimidine nucleotide precursors, together with a pharmaceuticallyacceptable carrier.

[0084] The compositions, depending on the intended use and route ofadministration, are manufactured in the form of a liquid, a suspension,a tablet, a capsule, a dragee, an injectable solution, or a suppository(see discussion of formulation below).

[0085] In another embodiment of the invention, the composition comprisesat least one pyrimidine nucleotide precursor and an agent which inhibitsthe degradation of uridine, such as an inhibitor of the enzyme uridinephosphorylase. Examples of inhibitors of uridine phosphorylase includebut are not limited to 5-benzyl barbiturate or 5-benzylidene barbituratederivatives including 5-benzyl barbiturate, 5-benzyloxybenzylbarbiturate, 5-benzyloxybenzyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate, 5-benzyloxybenzylacetyl-1-[(1-hydroxy-2-ethoxy)methyl]barbiturate, and 5-methoxybenzylacetylacyclobarbiturate,2,2′-anhydro-5-ethyluridine, and acyclouridine compounds, particularly5-benzyl substituted acyclouridine congeners including but not limitedto benzylacyclouridine, benzyloxybenzylacyclouridine,aminomethyl-benzylacyclouridine,aminomethylbenzyloxybenzylacyclouridine,hydroxymethyl-benzylacyclouridine, andhydroxymethyl-benzyloxybenzylacyclouridine. Furthermore, it is withinthe scope of the invention to utilize an inhibitor of uridinephosphorylase alone, without coadministration of a pyrimidine nucleotideprecursor, for the purpose of treating mitochondrial diseases orpathophysiologies associated with mitochondrial respiratory chaindysfunction.

[0086] Further embodiments of the invention comprise a pyrimidinenucleotide precursor combined with one or more other agents withprotective or supportive activity relative to mitochondrial structureand function. Such agents, presented with recommended daily doses inmitochondrial diseases include, but are not limited to, pyruvate (1 to10 grams/day), Coenzyme Q (1 to 4 mg/kg/day), alanine (1-10 grams/day),lipoic acid (1 to 10 mg/kg/day), carnitine (10 to 100 mg/kg/day),riboflavin (20 to 100 mg/day, biotin (1 to 10 mg/day), nicotinamide (20to 100 mg/day), niacin (20 to 100 mg/day), Vitamin C (100 to 1000mg/day), Vitamin E (200-400 mg/day), and dichloroacetic acid or itssalts. In the case of pyruvate, this active agent can be administered aspyruvic acid, pharmaceutically acceptable salts thereof, or pyruvic acidesters having an alcohol moiety containing 2 to 10 carbon atoms.

D. Therapeutic Uses of the Compounds and Compositions of the Invention

[0087] Diseases related to mitochondrial respiratory chain dysfunctioncan be divided into several categories based on the origin ofmitochondrial defects.

[0088] Congenital mitochondrial diseases are those related to hereditarymutations, deletions, or other defects in mitochondrial DNA or innuclear genes regulating mitochondrial DNA integrity, or in nucleargenes encoding proteins that are critical for mitochondrial respiratorychain function.

[0089] Acquired mitochondrial defects comprise primarily 1) damage tomitochondrial DNA due to oxidative processes or aging; 2) mitochondrialdysfunction due to excessive intracellular and intramitochondrialcalcium accumulation; 3) inhibition of respiratory chain complexes withendogenous or exogenous respiratory chain inhibitors; 4) acute orchronic oxygen deficiency; and 5) impaired nuclear-mitochondrialinteractions, e.g. impaired shuttling of mitochondria in long axons dueto microtubule defects.

[0090] The most fundamental mechanisms involved in acquiredmitochondrial defects, and which underlie pathogenesis of a variety offorms of organ and tissue dysfunction, include:

[0091] Calcium accumulation: A fundamental mechanism of cell injury,especially in excitable tissues, involves excessive calcium entry intocells, as a result of either leakage through the plasma membrane ordefects in intracellular calcium handling mechanisms. Mitochondria aremajor sites of calcium sequestration, and preferentially utilize energyfrom the respiratory chain for taking up calcium rather than for ATPsynthesis, which results in a downward spiral of mitochondrial failure,since calcium uptake into mitochondria results in diminishedcapabilities for energy transduction.

[0092] Excitotoxicity: Excessive stimulation of neurons with excitatoryamino acids is a common mechanism of cell death or injury in the centralnervous system. Activation of glutamate receptors, especially of thesubtype designated NMDA receptors, results in mitochondrial dysfunction,in part through elevation of intracellular calcium during excitotoxicstimulation. Conversely, deficits in mitochondrial respiration andoxidative phosphorylation sensitizes cells to excitotoxic stimuli,resulting in cell death or injury during exposure to levels ofexcitotoxic neurotransmitters or toxins that would be innocuous tonormal cells.

[0093] Nitric oxide exposure: Nitric oxide (^(˜)1 micromolar) inhibitscytochrome oxidase (Complex IV) and thereby inhibits mitochondrialrespiration (Brown G C, Mol. Cell. Biochem. 174:189-192, 1997);moreover, prolonged exposure to NO irreversibly reduces Complex Iactivity. Physiological or pathophysiological concentrations of NOthereby inhibit pyrimidine biosynthesis. Nitric oxide is implicated in avariety of neurodegenerative disorders, and is involved in mediation ofexcitotoxic and post-hypoxic damage to neurons.

[0094] Hypoxia: Oxygen is the terminal electron acceptor in therespiratory chain. Oxygen deficiency impairs electron transport chainactivity, resulting in diminished pyrimidine synthesis as well asdiminished ATP synthesis via oxidative phosphorylation. Human cellsproliferate and retain viability under virtually anaerobic conditions ifprovided with uridine and pyruvate (or a similarly effective agent foroxidizing NADH to optimize glycolytic ATP production).

[0095] Nuclear-mitochondrial interactions: Transcription ofmitochondrial DNA encoding respiratory chain components requires nuclearfactors. In neuronal axons, mitochondria must shuttle back and forth tothe nucleus in order to maintain respiratory chain activity. If axonaltransport is impaired by hypoxia or by drugs like taxol which affectmicrotubule stability, mitochondria distant from the nucleus undergoloss of cytochrome oxidase activity.

[0096] In the nervous system especially, mitochondrial respiratory chaindeficits have two generalizable consequences: 1) Delayed or aberrantdevelopment of neuronal circuits within the nervous system; and 2)accelerated degeneration of neurons and neural circuits, either acutelyor over a period of years, depending on the severity of themitochondrial deficits and other precipitating factors. Analogouspatterns of impaired development and accelerated degeneration pertain tonon-neural tissues and systems as well.

Mitochondrial Dysfunction and Pyrimidine Biosynthesis

[0097] Cells with severely damaged mitochondria (including totaldeletion of mitochondrial DNA, with a consequent shutdown of respiratorychain activity) can survive in culture if provided with two agents whichcompensate for critical mitochondrial functions: uridine and pyruvate.Uridine is required in vitro because a limiting enzyme for de novosynthesis of uridine nucleotides, dihydro-orotate dehydrogenase (DHODH),is coupled to the mitochondrial respiratory chain, via ubiquinone as aproximal electron acceptor, cytochrome c as an intermediate, and oxygenas a terminal electron acceptor (Loffler et al., Mol. Cell. Biochem.174:125-129, 1997). DHODH is required for synthesis of orotate, which isthen phosphoribosylated and decarboxylated to produce uridinemonophosphate (UMP). All other pyrimidines in cells are derived fromUMP. Cells from patients with mitochondrial disease due to defects inmitochondrial DNA require exogenous uridine in order to survive outsideof the milieu of the body, wherein pyrimidines, derived from other cellsor the diet, and transported via the circulation, are prima faciesufficient to support their viability (Bourgeron, et al. Neuromusc.Disord. 3:605-608, 1993). Significantly, intentional inhibition of DHODHwith drugs like Brequinar or Leflunomide results in dose-limitingcytotoxic damage to the hematopoietic system and gastrointestinalmucosa, in contrast to the predominant involvement of postmitotictissues like the nervous system and muscle in clinical mitochondrialdisease.

[0098] An important feature of the subject invention is the unexpectedresult that treatment of patients with mitochondrial disease caused by avariety of underlying molecular defects results in clinical improvementin a diverse assortment of symptoms in vivo in patients (Examples 1-4).It is significant and further unexpected that clinical benefit has beenobserved in patients with no detectable defects in the respiratory chaincomplexes (III and IV) that are involved in pyrimidine biosynthesis.

Treatment of Congenital Mitochondrial Cytopathies Mitochondrial DNADefects

[0099] A number of clinical syndromes have been linked to mutations ordeletions in mitochondrial DNA. Mitochondrial DNA is inheritedmaternally, with virtually all of the mitochondria in the body derivedfrom those provided by the oocyte. If there is a mixture of defectiveand normal mitochondria in an oocyte, the distribution and segregationof mitochondria is a stochastic process. Thus, mitochondrial diseasesare often multisystem disorders, and a particular point mutation inmitochondrial DNA, for example, can result in dissimilar sets of signsand symptoms in different patients. Conversely, mutations in twodifferent genes in mitochondrial DNA can result in similar symptomcomplexes.

[0100] Nonetheless, some consistent symptom patterns have emerged inconjunction with identified mitochondrial DNA defects, and thesecomprise the classic “mitochondrial diseases”, some of which are listedimmediately below. Nonetheless, an important aspect of the subjectinvention is the recognition that the concept of mitochondrial diseaseand its treatment with compounds and compositions of the inventionextends to many other disease conditions which are also disclosedherein.

[0101] Some of the major mitochondrial diseases associated withmutations or deletions of mitochondrial DNA include:

[0102] MELAS: (Mitochondrial Encephalomyopathy Lactic Acidemia, andStroke-like episodes.

[0103] MERRF: Myoclonic Epilepsy with “Ragged Red” (muscle) Fibers

[0104] NARP: Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa

[0105] LHON: Leber's Hereditary Optic Neuropathy

[0106] Leigh's Syndrome (Subacute Necrotizing Encephalomyopathy)

[0107] PEO: Progressive External Opthalmoplegia

[0108] Kearns-Sayres Syndrome (PEO, pigmentary retinopathy, ataxia, andheart-block)

[0109] Other common symptoms of mitochondrial diseases which may bepresent alone or in conjunction with these syndromes includecardiomyopathy, muscle weakness and atrophy, developmental delays(involving motor, language, cognitive or executive function), ataxia,epilepsy, renal tubular acidosis, peripheral neuropathy, opticneuropathy, autonomic neuropathy, neurogenic bowel dysfunction,sensorineural deafness, neurogenic bladder dysfunction, dilatingcardiomyopathy, migraine, hepatic failure, lactic acidemia, and diabetesmellitus.

[0110] In addition, gene products and tRNA encoded by mitochondrial DNA,many proteins involved in, or affecting, mitochondrial respiration andoxidative phosphorylation are encoded by nuclear DNA. In fact,approximately 3000 proteins, or 20% of all proteins encoded by thenuclear genome, are physically incorporated into, or associated with,mitochondria and mitochondrial functions, although only about 100 aredirectly involved as structural components of the respiratory chain.Therefore, mitochondrial diseases involve not only gene products ofmitochondrial DNA, but also nuclear encoded proteins affectingrespiratory chain function.

[0111] Metabolic stressors like infections can unmask mitochondrialdefects that do not necessarily yield symptoms under normal conditions.Neuromuscular or neurological setbacks during infection are a hallmarkof mitochondrial disease. Conversely, mitochondrial respiratory chaindysfunction can render cells vulnerable to stressors that wouldotherwise be innocuous.

[0112] As is demonstrated in the Examples, compounds and compositions ofthe invention are useful for treatment of a very broad spectrum of signsand symptoms in mitochondrial diseases with different underlyingmolecular pathologies. The broad applicability of the methods of theinvention are unexpected and set the compounds and compositions of theinvention apart from other therapies of mitochondrial disease that havebeen attempted e.g. Coenzyme Q, B vitamins, carnitine, and lipoic acid,which generally address very specific reactions and cofactors involvedin mitochondrial function and which are therefore useful only inisolated cases. However, such metabolic interventions with antioxidantsand cofactors of respiratory chain complexes are compatible withconcurrent treatment with compounds and compositions of the invention,and in fact are used to their best advantage in combination withcompounds and compositions of the invention.

Treatment of Neuromuscular Degenerative Disorders Friedreich's Ataxia

[0113] A gene defect underlying Friedreich's Ataxia (FA), the mostcommon hereditary ataxia, was recently identified and is designated“frataxin”. In FA, after a period of normal development, deficits incoordination develop which progress to paralysis and death, typicallybetween the ages of 30 and 40. The tissues affected most severely arethe spinal cord, peripheral nerves, myocardium, and pancreas. Patientstypically lose motor control and are confined to wheel chairs, and arecommonly afflicted with heart failure and diabetes.

[0114] The genetic basis for FA involves GAA trinucleotide repeats in anintron region of the gene encoding frataxin. The presence of theserepeats results in reduced transcription and expression of the gene.Frataxin is involved in regulation of mitochondrial iron content. Whencellular frataxin content is subnormal, excess iron accumulates inmitochondria, promoting oxidative damage and consequent mitochondrialdegeneration and dysfunction.

[0115] When intermediate numbers of GAA repeats are present in thefrataxin gene intron, the severe clinical phenotype of ataxia may notdevelop. However, these intermediate-length trinucleotide extensions arefound in 25 to 30% of patients with non-insulin dependent diabetesmellitus, compared to about 5% of the nondiabetic population.

[0116] Compounds and compositions of the invention are useful fortreating patients with disorders related to deficiencies or defects infrataxin, including Friedreich's Ataxia, myocardial dysfunction,diabetes mellitus and complications of diabetes like peripheralneuropathy. Conversely, diagnostic tests for presumed frataxindeficiencies involving PCR tests for GAA intron repeats are useful foridentifying patients who will benefit from treatment with compounds andcompositions of the invention.

Muscular Dystrophy

[0117] Muscular dystrophy refers to a family of diseases involvingdeterioration of neuromuscular structure and function, often resultingin atrophy of skeletal muscle and myocardial dysfunction. In the case ofDuchenne muscular dystrophy, mutations or deficits in a specificprotein, dystrophin, are implicated in its etiology. Mice with theirdystrophin genes inactivated display some characteristics of musculardystrophy, and have an approximately 50% deficit in mitochondrialrespiratory chain activity. A final common pathway for neuromusculardegeneration in most cases is calcium-mediated impairment ofmitochondrial function. Compounds and compositions of the invention areuseful for reducing the rate of decline in muscular functionalcapacities and for improving muscular functional status in patients withmuscular dystrophy.

Multiple Sclerosis

[0118] Multiple sclerosis (MS) is a neuromuscular disease characterizedby focal inflammatory and autoimmune degeneration of cerebral whitematter. Periodic exacerbations or attacks are significantly correlatedwith upper respiratory tract and other infections, both bacterial andviral, indicating that mitochondrial dysfunction plays a role in MS.Nitric oxide Depression of neuronal mitochondrial respiratory chainactivity caused by Nitric Oxide (produced by astrocytes) is implicatedas a molecular mechanism contributing to MS.

[0119] Compounds and compositions of the invention are useful fortreatment of patients with multiple sclerosis, both prophylactically andduring episodes of disease exacerbation.

Treatment of Disorders of Neuronal Instability Treatment of SeizureDisorders

[0120] Epilepsy is often present in patients with mitochondrialcytopathies, involving a range of seizure severity and frequency, e.g.absence, tonic, atonic, myoclonic, and status epilepticus, occurring inisolated episodes or many times daily.

[0121] In patients with seizures secondary to mitochondrial dysfunction,compounds and methods of the invention are useful for reducing frequencyand severity of seizure activity.

Treatment and Prevention of Migraine

[0122] Metabolic studies on patients with recurrent migraine headachesindicate that deficits in mitochondrial activity are commonly associatedwith this disorder, manifesting as impaired oxidative phosphorylationand excess lactate production. Such deficits are not necessarily due togenetic defects in mitochondrial DNA. Migraineurs are hypersensitive tonitric oxide, an endogenous inhibitor of Cytochrome c Oxidase. Inaddition, patients with mitochondrial cytopathies, e.g. MELAS, oftenhave recurrent migraines.

[0123] In patients with recurrent migraine headaches, compounds,compositions, and methods of the invention are useful for prevention andtreatment, especially in the case of headaches refractory to ergotcompounds or serotonin receptor antagonists.

[0124] As demonstrated in Example 1, compounds and compositions of theinvention are useful for treatment of migraines associate withmitochondrial dysfunction.

Treatment of Developmental Delay

[0125] Delays in neurological or neuropsychological development areoften found in children with mitochondrial diseases. Development andremodeling of neural connections requires intensive biosyntheticactivity, particularly involving synthesis of neuronal membranes andmyelin, both of which require pyrimidine nucleotides as cofactors.Uridine nucleotides are involved in activation and transfer of sugars toglycolipids and glycoproteins. Cytidine nucleotides are derived fromuridine nucleotides, and are crucial for synthesis of major membranephospholipid constituents like phosphatidylcholine, which receives itscholine moiety from cytidine diphosphocholine. In the case ofmitochondrial dysfunction (due to either mitochondrial DNA defects orany of the acquired or conditional deficits like exicitoxic or nitricoxide-mediated mitochondrial dysfunction described above) or otherconditions resulting in impaired pyrimidine synthesis, cellproliferation and axonal extension is impaired at crucial stages indevelopment of neuronal interconnections and circuits, resulting indelayed or arrested development of neuropsychological functions likelanguage, motor, social, executive function, and cognitive skills. Inautism for example, magnetic resonance spectroscopy measurements ofcerebral phosphate compounds indicates that there is globalundersynthesis of membranes and membrane precursors indicated by reducedlevels of uridine diphospho-sugars, and cytidine nucleotide derivativesinvolved in membrane synthesis (Minshew et al., Biological Psychiatry33:762-773, 1993).

[0126] Disorders characterized by developmental delay include Rett'sSyndrome, pervasive developmental delay (or PDD-NOS: “pervasivedevelopmental delay—not otherwise specified” to distinguish it fromspecific subcategories like autism), autism, Asperger's Syndrome, andAttention Deficit/Hyperactivity Disorder (ADHD), which is becomingrecognized as a delay or lag in development of neural circuitryunderlying executive functions.

[0127] Compounds and compositions of the invention are useful fortreating patients with neurodevelopmental delays involving motor,language, executive function, and cognitive skills. Current treatmentsfor such conditions, e.g. ADHD, involve amphetamine-like stimulants thatenhance neurotransmission in some affected underdeveloped circuits, butsuch agents, which may improve control of disruptive behaviors, do notimprove cognitive function, as they do not address underlying deficitsin the structure and interconnectedness of the implicated neuralcircuits.

[0128] Compounds and compositions of the invention are also useful inthe case of other delays or arrests of neurological andneuropsychological development in the nervous system and somaticdevelopment in non-neural tissues like muscle and endocrine glands.

Treatment of Neurodegenerative Disorders

[0129] The two most significant severe neurodegenerative diseasesassociated with aging, Alzheimer's Disease (AD) and Parkinson's Disease(PD), both involve mitochondrial dysfunction in their pathogenesis.Complex I deficiencies in particular are frequently found not only inthe nigrostriatal neurons that degenerate in Parkinson's disease, butalso in peripheral tissues and cells like muscle and platelets ofParkinson's Disease patients.

[0130] In Alzheimer's Disease, mitochondrial respiratory chain activityis often depressed, especially Complex IV (Cytochrome c Oxidase).Moreover, mitochondrial respiratory function altogether is depressed asa consequence of aging, further amplifying the deleterious sequelae ofadditional molecular lesions affecting respiratory chain function.

[0131] Other factors in addition to primary mitochondrial dysfunctionunderlie neurodegeneration in AD, PD, and related disorders. Excitotoxicstimulation and nitric oxide are implicated in both diseases, factorswhich both exacerbate mitochondrial respiratory chain deficits and whosedeleterious actions are exaggerated on a background of respiratory chaindysfunction.

[0132] Huntington's Disease also involves mitochondrial dysfunction inaffected brain regions, with cooperative interactions of excitotoxicstimulation and mitochondrial dysfunction contributing to neuronaldegeneration.

[0133] Compounds and compositions of the invention are useful forattenuating progression of age-related neurodegenerative diseaseincluding AD and PD.

Amyotrophic Lateral Sclerosis

[0134] One of the major genetic defects in patients with AmyotrophicLateral Sclerosis (ALS; Lou Gehrig's Disease; progressive degenerationof motor neurons, skeletal muscle atrophy, inevitably leading toparalysis and death) is mutation or deficiency in Copper-Zinc SuperoxideDismutase (SOD1), an antioxidant enzyme. Mitochondria both produce andare primary targets for reactive oxygen species. Inefficient transfer ofelectrons to oxygen in mitochondria is the most significantphysiological source of free radicals in mammalian systems. Deficienciesin antioxidants or antioxidant enzymes can result in or exacerbatemitochondrial degeneration. Mice transgenic for mutated SOD1 developsymptoms and pathology similar to those in human ALS. The development ofthe disease in these animals has been shown to involve oxidativedestruction of mitochondria followed by functional decline of motorneurons and onset of clinical symptoms (Kong and Xu, J. Neurosci.18:3241-3250, 1998). Skeletal muscle from ALS patients has lowmitochondrial Complex I activity (Wiedemann et al., J. Neurol. Sci156:65-72, 1998).

[0135] Compounds, compositions, and methods of the invention are usefulfor treatment of ALS, for reversing or slowing the progression ofclinical symptoms.

Protection Against Ischemia and Hypoxia

[0136] Oxygen deficiency results in both direct inhibition ofmitochondrial respiratory chain activity by depriving cells of aterminal electron acceptor for Cytochrome c reoxidation at Complex IV,and indirectly, especially in the nervous system, via secondarypost-anoxic excitotoxicity and nitric oxide formation.

[0137] In conditions like cerebral anoxia, angina or sickle cell anemiacrises, tissues are relatively hypoxic. In such cases, compounds of theinvention provide protection of affected tissues from deleteriouseffects of hypoxia, attenuate secondary delayed cell death, andaccelerate recovery from hypoxic tissue stress and injury.

Renal Tubular Acidosis

[0138] Acidosis due to renal dysfunction is often observed in patientswith mitochondrial disease, whether the underlying respiratory chaindysfunction is congenital or induced by ischemia or cytotoxic agentslike cisplatin. Renal tubular acidosis often requires administration ofexogenous sodium bicarbonate to maintain blood and tissue pH.

[0139] In Example 3, administration of a compound of the inventioncaused an immediate reversal of renal tubular acidosis in a patient witha severe Complex I deficiency. Compounds and compositions of theinvention are useful for treating renal tubular acidosis and other formsof renal dysfunction caused by mitochondrial respiratory chain deficits.

Age-Related Neurodegeneration and Cognitive Decline

[0140] During normal aging, there is a progressive decline inmitochondrial respiratory chain function. Beginning about age 40, thereis an exponential rise in accumulation of mitochondrial DNA defects inhumans, and a concurrent decline in nuclear-regulated elements ofmitochondrial respiratory activity.

[0141] de Grey (Bioessays, 19:161-167, 1998) discussed mechanismsunderlying the observation that many mitochondrial DNA lesions have aselection advantage during mitochondrial turnover, especially inpostmitotic cells. The proposed mechanism is that mitochondria with adefective respiratory chain produce less oxidative damage to themselvesthan do mitochondria with intact functional respiratory chains(mitochondrial respiration is the primary source of free radicals in thebody). Therefore, normally-functioning mitochondria accumulate oxidativedamage to membrane lipids more rapidly than do defective mitochondria,and are therefore “tagged” for degradation by lysosomes. Sincemitochondria within cells have a half life of about 10 days, a selectionadvantage can result in rapid replacement of functional mitochondriawith those with diminished respiratory activity, especially in slowlydividing cells. The net result is that once a mutation in a gene for amitochondrial protein that reduces oxidative damage to mitochondriaoccurs, such defective mitochondria will rapidly populate the cell,diminishing or eliminating its respiratory capabilities. Theaccumulation of such cells results in aging or degenerative disease atthe organismal level. This is consistent with the progressive mosaicappearance of cells with defective electron transport activity inmuscle, with cells almost devoid of Cytochrome c Oxidase (COX) activityinterspersed randomly amidst cells with normal activity, and a higherincidence of COX-negative cells in biopsies from older subjects. Theorganism, during aging, or in a variety of mitochondrial diseases, isthus faced with a situation in which irreplaceable postmitotic cells(e.g. neurons, skeletal and cardiac muscle) must be preserved and theirfunction maintained to a significant degree, in the face of aninexorable progressive decline in mitochondrial respiratory chainfunction. Neurons with dysfunctional mitochondria become progressivelymore sensitive to insults like excitotoxic injury. Mitochondrial failurecontributes to most degenerative diseases (especially neurodegeneration)that accompany aging.

[0142] Congenital mitochondrial diseases often involve early-onsetneurodegeneration similar in fundamental mechanism to disorders thatoccur during aging of people born with normal mitochondria. Thedemonstration disclosed in the Examples that compounds and compositionsof the invention are useful in treatment of congenital or early-onsetmitochondrial disease provides direct support for the utility ofcompounds and compositions of the invention for treatment of age-relatedtissue degeneration.

[0143] Compounds and compositions of the invention are useful fortreating or attenuating cognitive decline and other degenerativeconsequences of aging.

Mitochondria and Cancer Chemotherapy

[0144] Mitochondrial DNA is typically more vulnerable to damage than isnuclear DNA for several reasons:

[0145] 1. Mitochondrial DNA has a less sophisticated repair system thandoes nuclear DNA.

[0146] 2. Virtually all of the mitochondrial DNA strands encodeimportant proteins, so that any defect will potentially affectmitochondrial function. Nuclear DNA contains long regions that do notencode proteins, wherein mutations or damage are essentiallyinconsequential.

[0147] 3. Defective mitochondria often have a selection advantage overnormal, active ones during proliferation and turnover.

[0148] 4. Mitochondrial DNA is not protected by histones

[0149] Empirically, mitochondrial DNA damage is more extensive andpersists longer than nuclear DNA damage in cells subjected to oxidativestress or cancer chemotherapy agents like cisplatin due to both greatervulnerability and less efficient repair of mitochondrial DNA. Althoughmitochondrial DNA may be more sensitive to damage than nuclear DNA, itis relatively resistant, in some situations, to mutagenesis by chemicalcarcinogens. This is because mitochondria respond to some types ofmitochondrial DNA damage by destroying their defective genomes ratherthan attempting to repair them. This results in global mitochondrialdysfunction for a period after cytotoxic chemotherapy. Clinical use ofchemotherapy agents like cisplatin, mitomycin, and cytoxan is oftenaccompanied by debilitating “chemotherapy fatigue”, prolonged periods ofweakness and exercise intolerance which may persist even after recoveryfrom hematologic and gastrointestinal toxicities of such agents.

[0150] Compounds, compositions, and methods of the invention are usefulfor treatment and prevention of side effects of cancer chemotherapyrelated to mitochondrial dysfunction. This use of pyrimidine nucleotideprecursors for attenuation of cancer chemotherapy side effects isconceptually and biochemically distinct from toxicity reduction ofcytotoxic anticancer pyrimidine analogs, which is mediated thoughbiochemical competition at the level of nucleotide antimetabolites.

[0151] Example 5 illustrates the protective effect of oraltriacetyluridine in protecting against taxol-induced neuropathy.

[0152] Furthermore, hepatic mitochondrial redox state is one contributorto appetite regulation. Cancer patients often display “early satiety”,contributing to anorexia, weight loss, and cachexia. Energy metabolismis often seriously disrupted in cancer patients, with energy-wastingfutile cycles of hyperactive tumor glycolysis producing circulatinglactate, which is converted by the liver back to glucose.Chemotherapy-induced mitochondrial injury further contributes tometabolic disruption.

[0153] As indicated in Example 2, treatment with a compound of theinvention resulted in improved appetite in a patient with mitochondrialdisease.

Mitochondria and Ovarian Function

[0154] A crucial function of the ovary is to maintain integrity of themitochondrial genome in oocytes, since mitochondria passed on to a fetusare all derived from those present in oocytes at the time of conception.Deletions in mitochondrial DNA become detectable around the age ofmenopause, and are also associated with abnormal menstrual cycles. Sincecells cannot directly detect and respond to defects in mitochondrialDNA, but can only detect secondary effects that affect the cytoplasm,like impaired respiration, redox status, or deficits in pyrimidinesynthesis, such products of mitochondrial function participate as asignal for oocyte selection and follicular atresia, ultimatelytriggering menopause when maintenance of mitochondrial genomic fidelityand functional activity can no longer be guaranteed. This is analogousto apoptosis in cells with DNA damage, which undergo an active processof cellular suicide when genomic fidelity can no longer be achieved byrepair processes. Women with mitochondrial cytopathies affecting thegonads often undergo premature menopause or display primary cyclingabnormalities. Cytotoxic cancer chemotherapy often induces prematuremenopause, with a consequent increased risk of osteoporosis.Chemotherapy-induced amenorrhea is generally due to primary ovarianfailure. The incidence of chemotherapy-induced amenorrhea increases as afunction of age in premenopausal women receiving chemotherapy, pointingtoward mitochondrial involvement. Inhibitors of mitochondrialrespiration or protein synthesis inhibit hormone-induced ovulation, andfurthermore inhibit production of ovarian steroid hormones in responseto pituitary gonadotropins. Women with Downs syndrome typically undergomenopause prematurely, and also are subject to early onset ofAlzheimer-like dementia. Low activity of cytochrome oxidase isconsistently found in tissues of Downs patients and in late-onsetAlzheimer's Disease.

[0155] Appropriate support of mitochondrial function or compensation formitochondrial dysfunction therefore is useful for protecting againstage-related or chemotherapy-induced menopause or irregularities ofmenstrual cycling or ovulation. Compounds and compositions of theinvention, including also antioxidants and mitochondrial cofactors, areuseful for treating and preventing amenorrhea, irregular ovulation,menopause, or secondary consequences of menopause.

[0156] In Example 1, treatment with a compound of the invention resultedin shortening of the menstrual cycle. Since the patient was in apersistent luteal phase, her response indicates that the administeredpyrimidine nucleotide precursor reversed hyporesponsiveness to pituitarygonadotropins, which were presumably elevated to compensate for theovarian hyporesponsiveness of mitochondrial origin.

Diagnosis of Mitochondrial Disease

[0157] The striking response of patients with mitochondrial disease toadministration of compounds of the invention indicates that a clinicalresponse to a pyrimidine nucleotide precursor administered according tothe methods of the subject invention has diagnostic utility to detectpossible mitochondrial disease. Molecular diagnosis of molecular lesionsunderlying mitochondrial dysfunction is difficult and costly, especiallywhen the defect is not one of the more common mutations or deletions ofmitochondrial DNA. Since the compounds and compositions of the inventionare very safe when administered in accord with the methods of thesubject invention, therapeutic challenge with a pyrimidine nucleotideprecursor is an important diagnostic probe for suspected mitochondrialdisease, especially when used in conjunction with tests for variousaspects of mitochondrial dysfunction.

E. Administration and Formulation of Compounds and Compositions of theInvention

[0158] In the case of all of the specific therapeutic targets forpyrimidine nucleotide precursor therapy of mitochondrial disease,compounds of the invention are typically administered one to three timesper day. Acyl derivatives of uridine and cytidine are administeredorally in doses of 0.01 to 0.5 grams per kilogram of body weight perday, with variations within this range depending on the amount requiredfor optimal clinical benefit. Generally, optimum doses are between 0.05and 0.3 grams/kg/day, divided into two or three separate doses taken 6to 12 hours apart.

[0159] In the case of patients unable to receive oral medications,compounds of the invention, especially uridine, cytidine, and orotateesters can be administered, as required, by prolonged intravenousinfusion, delivering daily doses of 0.01 to 0.5 grams/kg/day.

[0160] The pharmacologically active compounds optionally are combinedwith suitable pharmaceutically acceptable carriers comprising excipientsand auxiliaries which facilitate processing of the active compounds.These are administered as tablets, suspensions, solutions, dragees,capsules, or suppositories. The compositions are administered forexample orally, rectally, vaginally, or released through the buccalpouch of the mouth, and may be applied in solution form by injection,orally or by topical administration. The compositions may contain fromabout 0.1 to 99 percent, preferably from about 50 to 90 percent of theactive compound(s), together with the excipient(s).

[0161] For parenteral administration by injection or intravenousinfusion, the active compounds are suspended or dissolved in aqueousmedium such as sterile water or saline solution. Injectable solutions orsuspensions optionally contain a surfactant agent such aspolyoxyethylenesorbitan esters, sorbitan esters, polyoxyethylene ethers,or solubilizing agents like propylene glycol or ethanol. The solutiontypically contains 0.01 to 5% of the active compounds.

[0162] Suitable excipients include fillers such as sugars, for examplelactose, sucrose, mannitol or sorbitol, cellulose preparations and/orcalcium phosphates, for example tricalcium phosphate or calcium hydrogenphosphate, as well as binders such as starch paste, using, for example,maize starch, wheat starch, rice starch or potato starch, gelatin,tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodiumcarboxymethyl cellulose and/or polyvinyl pyrrolidone.

[0163] Auxiliaries include flow-regulating agents and lubricants, forexample, silica, talc, stearic acid or salts thereof, such as magnesiumstearate or calcium stearate and/or polyethylene glycol. Dragee coresare provided with suitable coatings which, if desired, are resistant togastric juices. For this purpose, concentrated sugar solutions are used,which optionally contain gum arabic, talc, polyvinyl pyrrolidone,polyethylene glycol and/or titanium dioxide, lacquer solutions andsuitable organic solvents or solvent mixtures. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate are used. Dyestuffs or pigmentsare optionally added to the tablets or dragee coatings, for example, foridentification or in order to characterize different compound doses.

[0164] The pharmaceutical preparations of the present invention aremanufactured in a manner which is itself known, for example, by means ofconventional mixing, granulating, dragee-making, dissolving, orlyophilizing processes. Thus, pharmaceutical preparations for oral useare obtained by combining the active compound(s) with solid excipients,optionally grinding the resulting mixture and processing the mixture ofgranules, after adding suitable auxiliaries, if desired or necessary, toobtain tablets or dragee cores.

[0165] Other pharmaceutical preparations which are useful for oraldelivery include push-fit capsules made of gelatin, as well assoft-sealed capsules made of gelatin and a plasticizer such as glycerolor sorbitol. The push-fit capsules contain the active compound(s) in theform of granules which optionally are mixed with fillers such aslactose, binders such as starches and/or lubricants such as talc ormagnesium stearate, and, optionally stabilizers. In soft capsules, theactive compounds are preferably dissolved or suspended in suitableliquids such as fatty oils, liquid paraffin, or polyethylene glycols. Inaddition, stabilizers optionally are added.

[0166] Pharmaceutical preparations which are used rectally include, forexample, suppositories which consist of a combination of activecompounds with a suppository base. Suitable suppository bases are, forexample, natural or synthetic triglycerides, paraffin hydrocarbons,polyethylene glycols or higher alkanols. In addition, gelatin rectalcapsules which consist of a combination of the active compounds with abase are useful. Base materials include, for example, liquidtriglycerides, polyethylene glycols, or paraffin hydrocarbons. Inanother embodiment of the invention, an enema formulation is used, whichoptionally contains viscosity-increasing excipients likemethylcellulose, hydroxypropylmethylcellulose, carboxymethycellulose,carbopol, glycerine polyacrylates, or other hydrogels.

[0167] Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water soluble form, forexample, water soluble salts.

[0168] In addition, suspensions of the active compounds as appropriatein oily injection suspensions are administered. Suitable lipophilicsolvents or vehicles include fatty oils, for example, sesame oil, orsynthetic fatty acid esters, for example, ethyl oleate or triglycerides.Aqueous injection suspensions optionally include substances whichincrease the viscosity of the suspension which include, for example,sodium carboxymethylcellulose, sorbitol and/or dextran. The suspensionoptionally contains stabilizers.

F. Synthesis of the Compounds of the Invention

[0169] Acyl derivatives of cytidine and uridine are synthesizedtypically by acylation methods involving reaction of acid chlorides oracid anhydrides with cytidine or uridine.

[0170] The synthesis of 2′,3′,5′-tri-O-pyruvyluridine is shown inExample 6.

[0171] The following examples are illustrative, but not limiting of themethods and compositions of the present invention. Other suitablemodifications and adaptations of a variety of conditions and parametersnormally encountered in clinical therapy which are obvious to thoseskilled in the art are within the spirit and scope of this invention.

EXAMPLES Example 1 Treatment of a Multisystem Mitochondrial Disorderwith Triacetyluridine

[0172] A 29 year old woman with a partial Complex I deficiency, andwhose son was diagnosed with mitochondrial disease leading tostroke-like episodes, ataxia, and encephalopathy, presented with amultisystem mitochondrial disorder. Signs and symptoms includedhemiplegic/aphasic migraines, grand-mal seizures, neurogenic bowel andbladder dysfunction, requiring catheterization approximately four timesper day, dysphagia, autonomic and peripheral polyneuropathy producingpainful paresthesias, tachycardia/bradycardia syndrome, and poorfunctional capacity with inability to climb a flight of stairs withoutstopping to rest, and declining cognitive performance with episodes ofclouded sensorium and poor memory lasting hours to days.

[0173] After beginning treatment with 0.05 mg/kg/day of oraltriacetyluridine, and for a duration of at least 6 months, this patienthas not had seizures or migraines; her paresthesias related toperipheral neuropathy have resolved. She is able to void spontaneouslyon most days, requiring catheterization only once or twice per week.After 6 weeks of treatment with triacetyluridine, this patient was ableto walk a full mile, which she has been unable to do for the past twoyears because of inadequate functional capacity. Her episodes ofbradycardia during sleep and tachycardia during exertion have reduced infrequency; prior to treatment, tachycardia with a heart rate greaterthan 140 bpm occurred upon simple rise to stand, and after 6 weeks oftriacetyluridine, tachycardia occurred only on hills and stairs. Hersensorium has cleared and memory deficits have improved markedly.

[0174] During treatment, this patients' menstrual cycles shortened from4 weeks to two weeks, and she displayed a persistent luteal phase asevaluated by estradiol, progesterone, FSH and LH measurements. Afterseveral months, her cycle normalized to 4 weeks.

[0175] This patient demonstrates important features of the subjectinvention, in that 1) the compound of the invention caused improvementsin virtually all features of a complex multisystem disease related tomitochondrial dysfunction in a variety of tissues, and that 2) compoundsof the invention are unexpectedly useful for treating disease conditionsrelated to a partial Complex I deficiency, which affects a portion ofthe mitochondrial respiratory chain that is outside of the sequence ofelectron transfers directly involved in de novo pyrimidine biosynthesis.

[0176] The transient shortening of this patient's menstrual cycle isinterpreted as an improvement of ovarian function caused bytriacetyluridine in the face of excessive hormonal stimulation by whichthe neuroendocrine system was attempting to compensate for ovariandysfunction. Feedback between the ovaries and the hypothalamus led togradual normalization of cycle time.

Example 2 Treatment of Refractory Epilepsy

[0177] An 11 year old boy had refractory epilepsy since age 4.5,apparently due to a multiple mitochondrial DNA deletion syndrome. InDecember 1997, his condition deteriorated, including 2 admissions to anintensive care unit for crescendo epilepsy. Even with aggressiveregimens of standard anticonvulsive therapy, this patient was having 8to 10 grand-mal seizures per night, leaving him unable to attend schoolregularly or participate in sports activities. He also developed upperlip automaticity.

[0178] In the first three days after beginning treatment with oraltriacetyluridine (initially at a dose of 0.05 g/kg/day, andincrementally increased to 0.1 and then 0.24 g/kg/day over the course ofseveral weeks), there were no seizures, and involuntary lip movementsceased. There has subsequently been some recurrence of seizuresespecially during episodes of infection, though at a much lowerfrequency than prior to treatment with triacetyluridine. This patienthas been able to return to school and resume active participation insports. His appetite, cognitive function, and fine motor coordinationhave improved during therapy, resulting in improved academic performanceand in outstanding performance in sports activities like baseball.

Example 3 Treatment of Renal Tubular Acidosis

[0179] A 2 year-old girl, with Leigh's Syndrome (subacute necrotizingencephalopathy) associated with severe Complex I deficiency, displayedrenal tubular acidosis requiring intravenous administration of 25 mEqper day of sodium bicarbonate. Within several hours after beginningintragastric treatment with triacetyluridine at 0.1 g/mg/day, her renaltubular acidosis resolved and supplementary bicarbonate was no longerrequired to normalize blood pH. Triacetyluridine also resulted in rapidnormalization of elevated circulating amino acid concentrations, andmaintained lactic acid at low levels after withdrawal of dichloroacetatetreatment, which was previously required to prevent lactic acidosis.

Example 4 Treatment of Developmental Delay

[0180] A 4.5 year-old girl with epilepsy, ataxia, language delay, andfat intolerance, and dicarboxylic aciduria was treated withtriacetyluridine at a daily dose of 0.1 to 0.3 g/kg/day. Such treatmentresulted in a 50% decline in seizure frequency, improvement of ataxiaand motor coordination, restoration of dietary fat tolerance, andaccelerated development of expressive language capabilities.

Example 5 Prevention of Taxol-Induced Neuropathy

[0181] Peripheral neuropathy is a frequent, and often dose-limiting,side effect of important anticancer agents like cisplatin and taxol. Inthe case of taxol, sensory neuropathy occurs several days afteradminnstration. Taxol's mechanism of action involves stabilization ofmicrotubules, which is useful for treating cancers, but is deleteriousto peripheral neurons. Microtubule stabilization impairs axonaltransport of cellular components. Mitochondria shuttle between the cellbody and terminals of neurons, so that the expression of mitochondrialrespiratory chain components can be regulated by nuclear transcriptionfactors. During inhibition of mitochondrial shuttling, mitochondriadistant from the nucleus undergo decline in expression of respiratorychain subunits encoded by the mitochondrial genome, due to inadequateexposure to mtDNA transcription factors, resulting in regional neuronalenergy failure and other consequences of mitochondrial dysfunction.

[0182] Two groups of 10 mice each were treated with taxol, 21.6mg/kg/day for 6 consecutive days by intraperitoneal injection. Anadditional group of 10 mice received injections of vehicle alone. One ofthe groups of taxol-treated mice received oral triacetyluridine, 4000mg/kg b.i.d. Nine days after the initiation of taxol treatments,nociceptive sensory deficits were tested by determining tail-flicklatency after exposure of the tip of the tail to focused thermalradiation with an infrared heat lamp. In this system, delays in thetail-flick response to radiant heat correlate with sensory nervedeficits.

[0183] Group: Tail flick latency

[0184] Control (no taxol) 10.8±0.5 seconds

[0185] Taxol 16.0±3.1 seconds

[0186] Taxol+triacetyluridine 11.9±0.7 seconds

[0187] Taxol treatment impaired responses to painful stimuli as an indexof toxic sensory neuropathy. Oral triacetyluridine treatmentsignificantly attenuated taxol-induced alterations in tail-flicklatency.

Example 6 Synthesis of Uridine Pyruvate

[0188] A. The preparation of pyruvyl chloride was accomplished by thereaction of alpha, alpha-dichloromethyl methyl ether and pyruvic acidusing the procedure of Ottenheum and Man (Synthesis, 1975, p. 163).

[0189] B. Uridine (3.0 g, 12 mmol) was dried by toluene azeotrope undervacuum (3×), and then dissolved in DMF (20 mL) and pyridine (20 mL). Theresultant solution was cooled to −10 degrees C. and 6.0 mL of pyruvylchloride (produced in step A above) was added dropwise. The reactionmixture was stirred at room temperature under argon for 24 hours.Analysis by TLC (5% MeOH/CH₂Cl₂) showed the consumption of uridine. Thereaction mixture was evaporated to dryness and partitioned betweenCH₂Cl₂ and aqueous sodium bicarbonate. The organic layer was washed withwater, aqueous HCl (pH 3.0), and water; dried over sodium sulfate;concentrated; and purified using flash chromatography (silica gel, 5%MeOH/CH₂CH₂) to yield 1.4 g of uridine pyruvate, or2′,3′,5′-tri-O-pyruvyluridine.

[0190] While the present invention has been described in terms ofpreferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art. Therefore, it isintended that the appended claims cover all such equivalent variationsand modifications which come within the scope of the invention asclaimed.

1. A method for treating or preventing pathophysiological consequencesof mitochondrial respiratory chain dysfunction in a mammal comprisingadministering to said mammal in need of such treatment or prevention aneffective amount of a pyrimidine nucleotide precursor.
 2. A method as inclaim 1 wherein said respiratory chain dysfunction is caused by amutation, deletion, or rearrangement of mitochondrial DNA.
 3. A methodas in claim 1 wherein said respiratory chain dysfunction is caused bydefective nuclear-encoded protein components of the mitochondrialrespiratory chain.
 4. A method as in claim 1 wherein said respiratorychain dysfunction is caused by aging.
 5. A method as in claim 1 whereinsaid respiratory chain dysfunction is caused by administration ofcytotoxic cancer chemotherapy agents to said mammal.
 6. A method as inclaim 1 wherein said respiratory chain dysfunction is a deficit inmitochondrial Complex I activity.
 7. A method as in claim 1 wherein saidrespiratory chain dysfunction is a deficit in mitochondrial Complex IIactivity.
 8. A method as in claim 1 wherein said respiratory chaindysfunction is a deficit in mitochondrial Complex III activity.
 9. Amethod as in claim 1 wherein said respiratory chain dysfunction is adeficit in mitochondrial Complex IV activity.
 10. A method as in claim 1wherein said respiratory chain dysfunction is a deficit in mitochondrialComplex V activity.
 11. A method as in claim 1 wherein said pyrimidinenucleotide precursor is selected from the group consisting of uridine,cytidine, an acyl derivative of uridine, an acyl derivative of cytidine,orotic acid, an alcohol ester of orotic acid, or a pharmaceuticallyacceptable salt thereof.
 12. A method as in claim 11 wherein saidpyrimidine nucleotide precursor is an acyl derivative of cytidine.
 13. Amethod as in claim 11 wherein said pyrimidine nucleotide precursor is anacyl derivative of uridine.
 14. A method as in claim 11 wherein saidacyl derivative of uridine is 2′,3′,5′-tri-O-acetyluridine.
 15. A methodas in claim 11 wherein said acyl derivative of uridine is2′,3′,5′-tri-O-pyruvyluridine.
 16. A method as in claim 11 wherein thealcohol substitutent of said alcohol ester of orotic acid is ethanol.17. A method as in claim 11 wherein said pyrimidine nucleotide precursoris cytidine diphosphocholine.
 18. A method as in claim 11 wherein saidpyrimidine nucleotide precursor is administered orally.
 19. A method asin claim 11 wherein said pyrimidine nucleotide precursor is administeredin a dose of 0.01 to 1 gram per kilogram of bodyweight per day.
 20. Amethod as in claim 1 wherein said pathophysiological consequence ofmitochondrial respiratory chain dysfunction is a congenitalmitochondrial disease.
 21. A method as in claim 20 wherein saidcongenital mitochondrial disease is selected from the group consistingof MELAS, LHON, MERRF, NARP, PEO, Leigh's Disease, and Kearns-SayresSyndrome.
 22. A method as in claim 1 wherein said pathophysiologicalconsequence of mitochondrial respiratory chain dysfunction is aneurodegenerative disease.
 23. A method as in claim 22 wherein saidneurodegenerative disorder is Alzheimer's Disease.
 24. A method as inclaim 22 wherein said neurodegenerative disorder is Parkinson's disease.25. A method as in claim 22 wherein said neurodegenerative disorder isHuntington's Disease.
 26. A method as in claim 22 wherein saidneurodegenerative disorder is age-related decline in cognitive function.27. A method as in claim 1 wherein said pathophysiological consequenceof mitochondrial respiratory chain dysfunction is a neuromusculardegenerative disease.
 28. A method as in claim 27 wherein saidneuromuscular degenerative disease is selected from the group consistingof muscular dystrophy, myotonic dystrophy, chronic fatigue syndrome, andFriedreich's Ataxia.
 29. A method as in claim 1 wherein saidpathophysiological consequence of mitochondrial respiratory chaindysfunction is developmental delay in cognitive, motor, language,executive function, or social skills.
 30. A method as in claim 1 whereinsaid pathophysiological consequence of mitochondrial respiratory chaindysfunction is selected from the group consisting of epilepsy,peripheral neuropathy, optic neuropathy, autonomic neuropathy,neurogenic bowel dysfunction, sensorineural deafness, neurogenic bladderdysfunction, migraine, and ataxia.
 31. A method as in claim 1 whereinsaid pathophysiological consequence of mitochondrial respiratory chaindysfunction is selected from the group consisting of renal tubularacidosis, dilating cardiomyopathy, hepatic failure, and lactic acidemia.32. A method for preventing death or functional decline of post-mitoticcells in a mammal due to mitochondrial respiratory chain dysfunctioncomprising administration of an effective amount of a pyrimidinenucleotide precursor.
 33. A method as in claim 32 wherein saidpost-mitotic cells are neurons.
 34. A method as in claim 32 wherein saidpost-mitotic cells are skeletal muscle cells.
 35. A method as in claim32 wherein said post-mitotic cells are cardiomyocytes.
 36. A method fortreating developmental delay in cognitive, motor, language, executivefunction, or social skills in a mammal comprising administration of aneffective amount of a pyrimidine nucleotide precursor.
 37. A method asin claim 36 wherein said developmental delay is pervasive developmentaldelay or PDD-NOS.
 38. A method as in claim 36 wherein said developmentaldelay is Attention Deficit/Hyperactivity Disorder.
 39. A method as inclaim 36 wherein said developmental delay is Rett's Syndrome.
 40. Amethod as in claim 36 wherein said developmental delay is autism.
 41. Amethod for reducing side effects of cytotoxic cancer chemotherapy agentsby administering a pyrimidine nucleotide precursor, where said cytotoxicchemotherapy agent is not a pyrimidine nucleoside analog.
 42. A methodas in claim 41 wherein said side effects of cytotoxic cancerchemotherapy are selected from the group consisting of peripheralneuropathy, chemotherapy-induced menopause, chemotherapy-associatedfatigue, and depressed appetite.
 43. A method for diagnosingmitochondrial disease by administering a pyrimidine nucleotide precursorand assessing clinical improvement in signs and symptoms in a mammal.44. A compound selected from the group consisting of2′,3′,5′-tri-O-pyruvyluridine, 2′,3′-di-O-pyruvyluridine,2′,5′-di-O-pyruvyluridine, 3′,5′-di-O-pyruvyluridine,2′-O-pyruvyluridine, 3′-O-pyruvyluridine, and 5′-O-pyruvyluridine.
 45. Apharmaceutical composition comprising: (a) a pyrimidine nucleotideprecursor or a pharmaceutically acceptable salt thereof, and (b) pyruvicacid, a pharmaceutically acceptable salt thereof, or a pyruvic acidester.
 46. A method as in claim 1 further comprising administeringpyruvic acid, a pharmaceutically acceptable salt thereof, or a pyruvicacid ester.